Bioactivity in Rhododendron: A Systemic Analysis of Antimicrobial and Cytotoxic Activities and Their Phylogenetic and Phytochemical Origins

The exceptional diversity of the genus Rhododendron has a strong potential for identification, characterization, and production of bioactive lead compounds for health purposes. A particularly relevant field of application is the search for new antibiotics. Here, we present a comparative analysis of nearly 90 Rhododendron species targeted toward the search for such candidate substances. Through a combination of phytochemical profiles with antimicrobial susceptibility and cytotoxicity, complemented by phylogenetic analyses, we identify seven potentially antimicrobial active but non-cytotoxic compounds in terms of mass-to-charge ratios and retention times. Exemplary bioactivity-guided fractionation for a promising Rhododendron species experimentally supports in fact one of these candidate lead compounds. By combining categorical correlation analysis with Boolean operations, we have been able to investigate the origin of bioactive effects in further detail. Intriguingly, we discovered clear indications of systems effects (synergistic interactions and functional redundancies of compounds) in the manifestation of antimicrobial activities in this plant genus.

H y m e n a n t h e s ( 3 1 ) P e n t a n t h e r a ( 1 2 H y m e n a n t h e s ( 3 1 ) P e n t a n t h e r a ( 1 2    In addition, the m/z ratio and retention time of the 23 mostpredictive peaks as well as top 1%, 2% and 5% peaks (dark orange to bright orange) with respect to Cohen's κ correlation to antimicrobial activity across all 87 Rhododendron species are shown. The arrows denote the average m/z ratio and retention time, respectively.
Hymenanthes (31) Pentanthera (12) Rhododendron (38) Tsutsusi ( Figure S8 | The 25 most-and least-predictive LC-MS peaks with respect to Cohen's κ correlation for cytotoxicity towards IEC-6 cells across all 87 Rhododendron species. A sample is denoted as cytotoxic towards IEC-6 cells (green) if the MTT assay is significantly dropped. A compound, defined by an m/z ratio and retention time tuple, is denoted as present in a sample (gray) if its intensity is ≥ 10000. The * represents the significance of the p-values according to multiple testing correction by Benjamini-Hochberg (0.05).
Hymenanthes (31) Pentanthera (12) Rhododendron (38) Tsutsusi ( Figure S11 | The 50 most-predictive peak combinations with additive effects with respect to Cohen's κ correlation for antimicrobial activity across all 87 Rhododendron species. A sample is denoted as antimicrobial active (orange) if the radius of the agar diffusion assay is ≥ 0.6 cm. A compound, defined by an m/z ratio and retention time tuple, is denoted as present in a sample (gray) if its intensity is ≥ 10000. The * represents the significance of the p-values according to multiple testing correction by Benjamini-Hochberg (0.05). The combinations comprising one of the seven most-predictive peaks regarding antimicrobial activity and non-cytotoxicity at once are highlighted in bright orange.
Figure S12 | The 50 most-predictive peak combinations with alternative effects with respect to Cohen's κ correlation for antimicrobial activity across all 87 Rhododendron species. A sample is denoted as antimicrobial active (orange) if the radius of the agar diffusion assay is ≥ 0.6 cm. A compound, defined by an m/z ratio and retention time tuple, is denoted as present in a sample (gray) if its intensity is ≥ 10000. The * represents the significance of the p-values according to multiple testing correction by Benjamini-Hochberg (0.05). The combinations comprising one of the seven most-predictive peaks regarding antimicrobial activity and non-cytotoxicity at once are highlighted in bright orange. Figure S13 | Heatmap of the top 250 LC-MS peaks involved in the most-predictive alternative peak combinations regarding Cohen's κ correlation for antimicrobial activity across all 87 Rhododendron species (main panel). The upper right panel provides the overview of all 5,414 peaks included in the outperforming alternative peak combinations, namely κ ≥ 0.77. The combinations highlighted in red involve at least one of the 23 most-predictive peaks regarding the individual peak analysis, κ ≥ 0.68. The corresponding individual peak correlation coefficients are depicted in the thinner horizontal and vertical panels. Orange labels emphasize the two out of seven most-predictive peaks regarding antimicrobial activity and non-cytotoxicity.
Figure S14 | The 50 most-predictive LC-MS peak combinations with additive effects with respect to Cohen's κ correlation for cytotoxicity towards IEC-6 cells across all 87 Rhododendron species. A sample is denoted as cytotoxic towards IEC-6 cells (green) if the MTT assay is significantly dropped. A compound, defined by an m/z ratio and retention time tuple, is denoted as present in a sample (gray) if its intensity is ≥ 10000. The * represents the significance of the p-values according to multiple correction by Benjamini-Hochberg (0.05).
Figure S15 | The 50 most-predictive LC-MS peak combinations with alternative effects with respect to Cohen's κ correlation for cytotoxicity towards IEC-6 cells across all 87 Rhododendron species. A sample is denoted as cytotoxic towards IEC-6 cells (green) if the MTT assay is significantly dropped. A compound, defined by an m/z ratio and retention time tuple, is denoted as present in a sample (gray) if its intensity is ≥ 10000. The * represents the significance of the p-values according to multiple testing correction by Benjamini-Hochberg (0.05).